Provisional Patent Application Ser. No.: OC0000000063 11882
1. Field of Invention
The present invention relates to a filter membrane pusher construction and a system that utilizes such pusher to remove filter membranes with biological samples retained by filters from multi-well filtration plate generally and, more particularly, but not by way of limitation, to a novel non-destructive collection of filter-retained biological specimens including cells and tissues for their further examination and storage.
2. Background Art
Filtration plates containing microporous membrane filters are widely used in basic research and for high throughput biological testing. The incorporation of membrane filters into the bottom of 96-well plates allows exploiting the capabilities of 96-well formats including automated liquid handling and recording of multiple experimental data. Membrane-bottom microplates allow multiple sequential procedures (i.e. sample preparation, cell growth and fixation, and washing) to be performed rapidly within a single plate. Because of this, 96-well membrane-bottom plates (i.e. MultiScreen plates manufactured by Millipore Corporation and AcroWell plates manufactured by Pall Corporation) are widely used for high-throughput cell-based assays (lymphocytes, hybridomas) including large-scale screening of natural and/or synthesized products to determine their biological activity or toxicity. When plates are used to grow and collect cells on the filter membranes the latter need to be removed from the plates at the end of experiment (i.e. immunohistochemistry, in situ hybridization or laser microdissection) since cells attached to the membranes can be accurately investigated only using high-magnification microscopy which cannot be done on membranes attached to the plate. It is important that removal of filter membranes should not affect morphology of membrane-retained cells and tissues and cause their loss.
Another use of 96-well membrane-bottom microplates includes an enzyme-linked immunospot assays (elispot) to retain proteins released by cultured cells on the membrane and subsequently detect such proteins by staining them. Upon completing elispot experiments, membrane filters with stained protein spots need to be removed from the plate to quantify spots by high-magnification image analysis and for proper membrane storage (i.e. removed membranes can be laminated for protection). Since particles of the dye used to stain proteins are loosely attached to the filter membrane, the removal of membranes has to be done to avoid the detachment of dye particles from the membrane.
Filter punch constructions are disclosed in U.S. Pat. No. 4,974,462 to Rising and Montminy and in U.S. Pat. No. 5,146,794 to Rising et al. These punches allow removing filters from multi-well filtration plates. However, these filter punches utilize a sharp central piercing member that punctures the filter, and hence cannot be used for a non-destructive collection of biological specimens such as cells and tissues. In addition, those filter punches do not permit to flatten punched membranes onto adhesive film, since piercing member will be making a hole rather than evenly push on underlying materials.
Other punch devices are disclosed in U.S. Pat. No. 4,036,088 to Ruskin, U.S. Pat. No. 4,713,995 to Davi and U.S. Pat. No. 4,688,457 to Neilsen and Davi. These devices are designed for punching holes in paper. These punch devices cannot be used for punching filter membranes from multi-well filtration plates for two reasons. First, a paper insertion gap is too narrow to accommodate a filtration plate, and second, punches have concave punching surface and hence will not permit flattening of punched material onto underlying support.
Accordingly, it is a principal object of the present invention to provide means and method for a non-damaging removal of filter membranes with deposited specimens from filtration plates.
It is a further object of the invention to provide means and method for immediate transfer and flattening of removed filter membranes onto adhesive support for retention.
It is a further object of the invention to provide means and method for simultaneous removal of all filter membranes from a plurality of wells and simultaneous transfer of all removed filter membranes onto adhesive support.
Other objects of the present invention, as well as particular features and advantages thereof, will be elucidated in, or apparent from, the following description and the accompanying drawings.
The present invention provides a pusher and a removing system for non-destructive removal of filters with retained biological samples from multi-well filtration plates. The pusher comprises a holder and a plurality of pushpins extending from the holder and perpendicular to it. Each pushpin has a round flat face-leading surface to provide an even distribution of pressure over the surface of underlying filter membrane and covering the most of the section of the filter membrane which is not attached to the well. Pushpins are aligned to match corresponding wells in the multi-well filtration plate. After the pusher is plugged into the multi-well filtration plate such assembly is inserted into the opening of the removal device where removal of filter membranes and their immediate transfer onto adhesive support will take place. The removing device comprises a base member and represents lower (steady) and upper (moving) platforms between which the aforementioned assembly of plate and pusher will be placed. Using a lever connected to the upper platform, the operator applies pressure over the pusher plate forcing its multiple pins into corresponding wells. During the operation, pressure pins of the pusher plate first detach the membranes from the wells and then these pressure pins press membranes against the adhesive support glued to the bottom of the membrane-backed plate. After completing the membrane-removing procedure, the membrane-backed plate with plugged-in pusher plate is removed from the membrane-removing device. After that adhesive support with individual membranes attached to it is pealed of the membrane-backed plate. This invention provides researchers who use membrane-backed plates with the tool that facilitates collection, analysis and storage of multiple membranes after completing the experiment. This system is alternative to laborious and tedious single-pin manual membrane removal procedure that cause damage to membranes which, in turn, results in loss of valuable experimental data. In addition, the system is user-friendly and does not require any specific training on the part of the operator. Furthermore, our system allows the shortening of membrane removal time and thus makes this a device of choice for researchers who run high-throughput assays using membrane-backed plates.